Murine resident macrophages produce an abundance of eicosanoids, whereas elicited macrophages produce lesser quantities of eicosanoids in general, and leukotriene C4 (LTC4) and prostacyclin (PGI2) in particular. Macrophage precursors derived from bone marrow cells produce even smaller amounts. We postulated that these differences may be regulated by substances found in the microenvironment of the cell, which may alter arachidonate release from phospholipid and its subsequent metabolism to eicosanoids. To examine if inherent differences in phospholipid availability contributed to the observed differences in eicosanoid synthesis among these three groups of macrophages, we monitored uptake and release of arachidonic acid (AA) in resident and elicited peritoneal macrophages and in bone marrow-derived macrophages (BMDM). Although differences existed in the extent of arachidonate release (37% vs. 22% vs. 27% release), the differences were not enough to explain the much larger differences in eicosanoid production. We therefore determined whether the AA cascade enzymes, including phospholipase A2 (PLA2) were intact by adding exogenous AA to the three cell types. PGI2 synthesis was not significantly increased in either elicited or BMDM. However, the enzymes necessary for LTC4 production appeared intact in elicited cells but not in BMDM. To further characterize the differences in eicosanoid synthesis between resident and elicited peritoneal macrophages and BMDM, we determined if a variety of exogenous substances [growth factors, cytokines, and noninflammatory and inflammatory peritoneal lavage fluid (NPLF and IPLF)] could enhance the production of LTC4 and PGI2 in those macrophage groups. The addition of granulocyte-macrophage colony stimulating factor (GM-CSF) slightly increased LTC4 production by BMDM and elicited macrophages. In contrast, NPLF increased the production of both LTC4 and PGI2 from BMDM, while IPLF had no effect. A similar effect of NPLF was seen on LTC4 (but not PGI2) production from elicited peritoneal cells, while IPLF decreased both LTC4 and PGI2 production from resident peritoneal macrophages. These studies indicate that substances found in the peritoneum of mice can enhance or diminish the production of LTC4 and PGI2 from the macrophage. This regulation appears to depend on the inflammatory state of the peritoneum.